Bacterial pathogens must adapt to changing environments in vivo in order to persist during infection within their host. Identifying genes that are functionally required for survival and fitness of the pathogen within the host helps to better understand pathogenesis, inform genome annotations, and guide the development of new therapeutic approaches. Tn-seq is a powerful genetic approach that allows researchers to identify en masse genes that are important for fitness in vivo using relevant models of infection. Streptococcus pyogenes (Group A Streptococcus, GAS) is a strict human pathogen and is listed among the top 10 causes of acutely life threatening bacterial infections worldwide, causing a wide array of diseases from self-limiting superficial infections of the skin & throat to severe invasive diseases of soft tissues & sterile sites. Our group has established the tools and experience to perform genome-wide genetic screens in GAS using the clinically relevant M1T1 strain 5448, and we have now completed the first in vivo Tn-seq of GAS infection in a murine model of localized GAS soft tissue infection to identify genes necessary for fitness during ulcerative lesion formation. Amongst our dataset, we identified two unannotated genes (called here subcutaneous fitness genes scfA and scfB) that were extremely important for GAS fitness in the lesion at both 24 and 48 hours post infection. Both genes are uncharacterized in GAS, but are predicted to encode membrane-associated proteins and are highly conserved among Firmicutes. The only study on scfAB homologs in the literature found them to be critical for the acid stress response and biofilm formation in S. mutans; suggesting that ScfAB functioned as a membrane permease complex. Our preliminary studies found that defined GAS M1T1 5448 mutants in scfA and scfB were outcompeted by wild type in vivo and were attenuated for survival following single strain infection in the soft tissue model. We hypothesize that the scfAB genes play an integral role in enhancing adaptation of GAS during murine soft tissue infection, and potentially in other host environments, through an as yet unknown mechanism that might involve membrane transport. The primary goal in this R21 proposal is to explore the role of ScfA and ScfB in GAS physiology and pathogenesis, with the expectation that this work will advance our understanding of how they might contribute to other important G+ pathogens. Thus, we seek to establish the contribution of scfAB to the pathophysiology of GAS via two aims: 1) Establish the localization, regulation and function of scfAB in GAS cell physiology, and 2) Investigate the impact of scfAB on GAS pathogenesis and colonization.